Method of purifying dry-cleaning solvent

ABSTRACT

PCT No. PCT/DK89/00308 Sec. 371 Date Sep. 5, 1990 Sec. 102(e) Date Sep. 5, 1990 PCT Filed Dec. 22, 1989 PCT Pub. No. WO90/07606 PCT Pub. Date Jul. 12, 1990.Contaminants containing non-polar neutral lipid are removed from a solvent that has been used for dry cleaning by placing used solvent in contact with a lipase, which is stable and exhibits an activity in the solvent, or with an immobilized product of said lipase, and with an adsorbent.

TECHNICAL FIELD

The present invention relates to a method of removing contaminants,especially neutral lipid contaminants, from a solvent which has beenused for dry cleaning, by using a lipase.

BACKGROUND ART

Solvents used for dry cleaning are commonly re-used after removal ofcontaminants by filtration and adsorption. However, non-polar neutrallipid contaminants are poorly adsorbed and are highly soluble in thesolvent, and they are therefore difficult to remove.

It is the purpose of the present invention to provide a purificationmethod that can readily remove contaminants containing non-polar neutrallipid from a solvent that has been used for dry cleaning.

STATEMENT OF THE INVENTION

The above-mentioned problem is solved by a purification methodcharacterized by placing used solvent in contact with a lipase, which isstable and exhibits an activity in the solvent, or with an immobilizedproduct of said lipase, and with an adsorbent

DETAILED DESCRIPTION OF THE INVENTION

Lipase produced by a microorganism belonging to the genus Candida,Humicola, Pseudomonas or Mucor or lipases produced by a transformantobtained by inserting the structural gene for said lipase into anothermicroorganism can be used advantageously.

Generally, enzymes do not show their activities in organic solventsbecause they undergo denaturation. However, it was surprisingly foundthat some lipases show activity even in dry cleaning solvents Forexample, lipases derived from Candida antarctica, Humicola lanuoinosaand Pseudomonas fluorescens show activity even in dry cleaning solvents.The present invention is based on this discovery. Neutral lipidcontaminants dissolved in solvents are decomposed by lipases and theresulting fatty acid and glyceride products are easily adsorbed on theadsorbents

The lipase can be added in the form of an aqueous solution to thesolvents used for dry cleaning, but may possibly recontaminate theclothes to be cleaned Consequently, it is desirable to use the lipase inthe form of immobilized lipase. Any known method can be used for theimmobilization of the lipase. For example, the immobilization can bedone by gel entrapment e.g. in polyacrylamide or alginate, by adsorptione.g. on silica or alumina, by crosslinking with glutaraldehyde, or byadsorption on ion exchange resin. (The details for the immobilizationare described, for example, in "Immobilized enzymes" written by Dr.Ichiro Chibata, published in 1975 by Kodansha Ltd.).

The contact between immobilized lipase and solvent is convenientlyeffected by use of a cartridge wherein the immobilized lipase isretained, while the solvent is allowed to flow through. Optionally, thecartridge may contain an adsorbent together with the immobilized lipase.Filter paper, filter cloth or other porous sheet material may be used toretain the immobilized lipase. The cartridge may allow the solvent to bepumped through; e.g. it may be cylindrical and have an inner tube and anouter, annular speace for solvent inlet and outlet. Or the cartridge mayconsist of a bag of porous material containing the immobilized lipase(and, optionally adsorbent), suited for dropping into a solvent tank.

The lipase may be a lipase produced by the original microorganism, or itmay be a lipase produced by a transformant produced by inserting thestructural gene for a lipase into another microorganism. Said genetransformant can be produced by generally known methods. Details aredescribed, for example, in Idenshi Kogaku (Genetic Engineering), Volume8 of Biseibutsu-gaku Kiso Koza (Basic Microbiological Seminars), editedby Tadahiko Ando and Kenji Sakaguchi, published in 1987 by KyoritsuShuppan Co., Ltd. and in Idenshi Kenkyuho (Genetic Laboratory Methods)II, Volume 1 of ZokuSeikagaku Jikken Koza (Sequel of BiochemicalExperiment Seminar), edited by Japan Society of Biochemistry, publishedin 1986 by K. K. Tokyo Kagaku Dojin.

Some lipases which can be used for the practice of the present inventionare commercially available. Some examples of suitable lipases are listedbelow.

(A) Immobilized Mucor miehei lipase (Lipozyme™, product of Novo-NordiskA/S, Denmark).

(B) Lipase derived from Humicola lanuginosa (SP-400, product ofNovo-Nordisk A/S, Denmark). This lipase is described in the JP-A63-68697.

(C) Lipase derived from Candida antarctica, described in WO 88/02775.

(D) Lipase derived from Pseudomonas fluorescens (product of AmanoPharmaceutical Co., Ltd.).

(E) Lipase derived from Pseudomonas cepacia, described in JP-A 62-34997,and the immobilized form of this enzyme (product of Novo-Nordisk A/S,Denmark), described in WO 89/01032.

The dry cleaning solvent may be e.g. trichloro-ethane,tetrachloro-ethylene, a hydrocarbon solvent (e.g. gasoline No. 5) or afluorinated hydrocarbon (e.g. Freon F-11 or F113). The method of theinvention is effective with any of these solvents.

The method for putting the solvent used for dry cleaning into contactwith lipase or immobilized lipase and with adsorbent is not speciallyrestricted; the lipase or immobilized lipase can be used in aconventional method for adsorption using adsorbent A particularlyadvantageous method consists in throwing into a tank with solvent acartridge in which immobilized lipase alone or in combination withadsorbent is contained in a vessel.

The adsorbents to be used for the present invention are not especiallyrestricted. For example, activated carbon powder and alumina silica gelcan be used advantageously.

The amount of lipase or immobilized lipase is preferably within therange of 1-50 weight percent of the solvent, and the amount of adsorbentis preferably within the range of 10-60 weight percent of the solvent.The ratio between the lipase or immobilized lipase and the adsorbent ispreferably within the range of 1/1-1/100.

The purification method of the present invention will be explainedhereinafter by way of working examples.

EXAMPLE 1

An excess of lipase derived from Humicola lanuoinosa (SP 400, a productof Novo-Nordisk A/S) was added to 20 g of porous ceramic (a product ofShowa Kogyo Co., Ltd.) in such a manner that it was entirely submergedinto the lipase solution and the mixture was left standing overnight soas to adsorb and immobilize the enzyme into the ceramic. The excessenzyme was removed by repeated washing with water and finally theceramic was washed with ethanol, which was dried in vacuo, by which theimmobilized enzyme could be obtained. The moisture content in theimmobilized enzyme thus obtained, determined with an infraredhygrometer, was less than 0.5 weight percent. By determination with asynthetic substrate it was found that the activity of the immobilizedenzyme was 40.9 units per gram.

The activity of the immobilized enzyme was determined in the followingmanner. Into 3 ml of 50 mM Tris hydrochloric acid buffer (pH 8.5), 0.45ml of an ethanolic solution containing 10 mg of immobilized enzyme and 1mmol of para-nitrophenyl capronate was added, and the increase in theabsorbance was determined with a wavelength of 400 nanometer. One unitwas defined herein as the amount of enzyme which can release 1 micromolof para-nitrophenol in one minute.

The hydrolysis of olive oil in trichloroethane was conducted using theabove-mentioned immobilized enzyme. In this process, water should besupplied in a certain amount because it is one of the substratesinvolved. For this reason, the immobilized enzyme was hydrated overnightin advance by adding 55 weight percent of water. The test sample usedwas 10 g of olive oil in 70 ml of trichloroethane, and 10 ml each of themixture was distributed into 4 test tubes with tight stoppering. Intothe test tubes Nos. 3 and 4, one gram each of the immobilized enzyme wasadded, and the mixtures were each reacted for 19 hours with gentlestirring. The test tubes Nos. 1 and 2 were left standing intact ascontrols. At 19 hours, 2 g each of a commercial adsorbent for drycleaning (Alumina silica gel "Sekard", a product of Shinagawa Kasei Co.,Ltd., Japan) was added into the test tubes Nos. 2 and 4. And thestirring was further continued for another 3 hours. One ml was collectedfrom each of these test tubes, and 10 ml each of chloroform containing15 weight percent of lithocolic acid as internal standard was added. Thecontents of triglycerides (TG), diglycerides (DG) and fatty acids inthese test samples were quantitatively assayed with Iatroscan (a productof Iatron Co., Ltd., Japan).

The results were as illustrated in FIG. 1. The number 1 in the chartrepresents the intact test sample (for comparison), while the numbers 2,3 and 4 represent the test samples with only adsorbent (for comparison),the sample with only enzyme (for comparison), and the sample treatedwith both immobilized enzyme and adsorbent (sample according to thepresent invention), respectively. As clearly seen in FIG. 1,triglycerides cannot be adsorbed at all when only adsorbent is added tothe olive oil solution When immobilized enzyme was added, the amounts ofdiglycerides and fatty acids were found to increase, showing thathydrolysis proceeded. The relatively low reaction rate can possibly beattributed to equilibrium being reached because of the low solubilitiesof diglycerides and fatty acids (as the reaction products) intrichloroethane. When both adsorbent and immobilized enzyme were used,the reaction products were adsorbed and the equilibrium moved to theside of hydrolysis, so that the hydrolysis remarkably advanced and thelipids in the solvent were remarkably reduced.

As clearly seen from this example, the method claimed by the presentinvention enables the decomposition of neutral lipids retained in useddry cleaning solvent into fatty acids and diglycerides, and thesereaction products can be easily adsorbed on an adsorbent, which can beeasily removed. Especially to be noted is that the adsorbent and lipasework with each other in a synergistic manner. More specifically, whenthe adsorbent and lipase are used together, the reaction products areadsorbed and the equilibrium moves to the side of hydrolysis, wherebythe hydrolysis is enhanced and the amount of lipid in the solvent isremarkably reduced.

FIG. 1 illustrates the effects of the method claimed by the presentinvention (using immobilized lipase and the adsorbent) for removal ofneutral lipids.

EXAMPLE 2

Olive oil degradation in trichloro ethylene tried using Lipozyme.Lipozyme was hydrated with 60% water (w/w) overnight. One gram of oliveoil was dissolved in 100 ml trichloro ethylene. 20 ml of this solutionwas taken into each of two flasks. One gram of Lipozyme was added to oneflask and 1 g each of Lipozyme and adsorbent were added to the other.The reaction mixture was kept for 24 hours One ml aliquot was taken fromthe reaction mixture, and 10 ml chloroform containing 0.5% (w/v)lithocholic acid was added as an internal standard. The amount oftriglyceride, diglyceride and fatty acid in each sample was analyzed byIatroscan. The result is shown in the table.

    ______________________________________                                                     TG (mg)  DG (mg)  FA (mg)                                        Sample         (in 10 ml sample)                                              ______________________________________                                        No treatment   68.4       0        33.4                                       Enzyme treatment                                                                             36.4       0        46.5                                       Enzyme + adsorbent                                                                           18.1       0        34.6                                       ______________________________________                                    

The reason why the amount of TG was decreased and FA was increased inthe enzyme treated sample compared with the non-treated sample is thatthe anion exchange resin which is used as a carrier of Lipozyme adsorbedsome FA produced by hydrolysis Total amount of TG and FA was decreasedto approx. 80% compared to the non-treated sample. When adsorbent wasadded together with enzyme the total amount was reduced to approx. 50%.

EXAMPLE 3

Olive oil degradation in 1,1,2-trichloro-1,2,2trifluoroethane (freon113) was tried using Lipase P "Amano". One gram of olive oil wasdissolved in 50 ml freon 113. One ml of 5% solution of Lipase P "Amano"and 4 g of adsorbent were added to the 20 ml of olive oil/freon 113. Thereaction mixture was kept for 16 hours. One ml aliquot was taken fromthe reaction mixture, and 10 ml chloroform containing 0.5% (w/v)lithocholic acid was added as an internal standard. The amount oftriglyceride, diglyceride and fatty acid in each sample was analyzed byIatroscan. An untreated sample was analyzed in the same way. It wasfound that TG had completely disappeared after the treatment, and theamount of FA was less than 10% of original amount of TG and FA.

I claim:
 1. A method for removing non-polar lipid contaminants from anorganic dry cleaning solvent comprising placing the solvent in contactwith (a) a lipase which is stable and exhibits an activity in thesolvent or an immobilized product of said lipase and (b) an adsorbent.2. The method according to claim 1, wherein the dry cleaning solvent istrichloroethane, tetrachloroethylene, a hydrocarbon solvent or afluorinated hydrocarbon.
 3. The method according to claim 2, wherein thedry cleaning solvent is gasoline no. 5, Freon F-11 or Freon F-113. 4.The method according to claim 1, wherein the removal of the contaminantsis effected by placing the solvent in contact with a cartridgecontaining the immobilized lipase without the adsorbent and subsequentlyplacing the solvent in contact with the absorbent.
 5. The methodaccording to claim 1, wherein the removal of the contaminants iseffected by placing the solvent in contact with a cartridge containingboth the immobilized lipase and the adsorbent.
 6. The method accordingto claim 1, wherein the lipase is produced by cultivation of amicroorganism belonging to the genus Candida, Humicola, Pseudomonas orMucor or by cultivation of a genetic recombinant of the lipase.
 7. Themethod according to claim 6, wherein the lipase is derived from Candidaantarctica, Humicola lanuginosa or Pseudomonas fluorescens.
 8. Themethod according to claim 1, 6 or 7, wherein the lipase is immobilizedby gel entrapment, by adsorption on silica or alumina, by cross-linkingwith glutaraldehyde or by adsorption on an ion exchange resin.
 9. Themethod according to claim 1, wherein the adsorbent is carbon powder oralumina silica gel.